1. Field of the Invention
The present invention relates generally to the fields of immunology and tumor therapy. More particularly, it concerns homoconjugates of antibodies which arrest cell growth and/or signal apoptosis in the tumor cells. The components of these homoconjugate antibodies can comprise a wide variety of antibodies but often and surprisingly do not require an Fc region to function and importantly activate fewer, or even no, undesired immunological reactions.
2. Description of Related Art
During the past two decades, a variety of monoclonal antibodies (MAbs) have been selected for clinical use based on their effector functions and the most encouraging results have emerged from the treatment of NHL (Hjekman et al., 1991; Press et al., 1987; Meeker et al., 1985; Waldmann, 1992; Hale et al., 1988; Dyer et al., 1989; Hamblin et al., 1987; Brown et al., 1989; Rankin et al., 1985) and, in particular, when radioimmunoconjugates were used (Kaminski et al., 1993; Press et al., 1993). Two examples are the epithelial cell-reactive MAb, 17.1A (Herlyn and Koprowski, 1982; Riethmüller et al., 1994) and the lymphoma reactive MAb, CAMPATH-1 (Dyer et al., 1989; Hale et al., 1988). In this regard, there is considerable experimental (Hooijberg et al., 1995a; Hooijberg et al., 1995b), and some clinical (Riethmüller et al., 1994; Hale et al., 1988) evidence to indicate that effector functions play an important role in the anti-tumor activity of MAbs. Those MAbs which do not fix complement or mediate antibody-dependent cell mediated cytotoxicity (ADCC) have been converted into useful ones by chimerization (Morrison and Oi, 1989; Adair, 1992), by generating switch variants (Hale et al., 1985; Kaminski et al., 1986; Denkers et al., 1995) or by preparing cytotoxic immunoconjugates (Dillman, 1994; Ghetie et al., 1996b; Hellström et al., 1995).
It has been demonstrated that the crosslinking of mIg on human Daudi cells initiates a cascade of signals leading to the induction of both apoptosis and CCA (Racila et al., 1996). Using antisense oligonucleotides, it was shown that the mIg-associated Lyn tyrosine kinase was required for anti-Ig mediated CCA but not for the induction of apoptosis (Racila et al., 1995). It was also shown that Lyn was critical for the induction of CA by anti-CD19 (Racila et al., 1995). These results suggest that the signaling pathways leading to CCA and apoptosis might bifurcate at an early stage of BCR crosslinking.
In attempting to further distinguish the different signaling pathways, studies were conducted to understand signal transduction initiated by crosslinking BCRs. Recent evidence supports a mechanism whereby TCR-induced apoptosis is dependent on Fas/Fas ligand interactions. Thus, crosslinking TCRs results in transient upregulation of the Fas ligand in T-lymphoma cells (Dhein et al., 1995; Brunner et al., 1995; Ju et al., 1995). Apoptosis induced by T cell receptor/T cell receptor (TCR) crosslinking is markedly inhibited by either anti-Fas F(ab′)2 fragments (which are not cytotoxic) or soluble Fas-Fc (Dhein et al., 1995; Brunner et al., 1995; Ju et al., 1995). These results indicate that in T-lymphoma cells, apoptosis induced by TCR activation results from the induction of the expression of the Fas ligand and its interaction with Fas resulting in the activation of the Fas signaling pathway.
In contrast to T cells, crosslinking of membrane IgM on Daudi cells (which constitutively express Fas) did not induced synthesis of Fas ligand (Racila et al., 1996). Furthermore, a noncytotoxic fragment of anti-Fas that blocked T cell receptor-mediated apoptosis did not block anti-μ induced apoptosis. Hence, in the B lymphoma cells, Daudi, apoptosis induced by signaling via IgM is not mediated by the Fas ligand. Similar results were obtained using a murine lymphoma (Scott et al., 1996). More recent studies suggest that the signaling is related to TNFα and TNFβ which are the only members of the TNF family which are upregulated in Daudi cells after crosslinking IgM. These studies suggest that IgM and CD19 may utilize different signaling pathways and that the activation pathways in Daudi cells are different from those induced via TCRs in T lymphoma cells.
Hence, like anti-CD40 (Marches et al., 1996), anti-CD19 enhanced the apoptosis induced by anti-μ. This finding is relevant for clinical strategies because it is obviously more desirable to kill a tumor cell than to induce CCA. The combination of anti-μ and anti-CD19 was studied in SCID/Daudi mice (where there is no serum IgM). Using multiple titrations of the two agents in the animals with a relatively long read-out period of 150 days. (Anti-μ will not be useful as a therapeutic agent in normal mice and humans, but is useful as a demonstration model.) In studying several MAbs which react with these molecules, it was found that none signaled as well as the anti-CD19 (HD37). It is postulated that either the target molecules could not readily mediate signals when bound to MAbs and/or that the ability of the MAbs to crosslink their target molecules was poor.
The therapeutic potential of MAbs has not been fully realized because there has been no paradigm for predicting which properties of the MAb are essential and how MAbs interact with other therapeutic modalities. For example, until recently, it was believed that the antitumor activity of a MAb was due solely to its conventional immunological effector mechanisms (i.e., ADCC, C′ fixation) (Dyer et al., 1989; Hale et al., 1984). Although this is true in certain instances, there is accumulating evidence that the antitumor activity of many MAbs is due to their ability to signal growth arrest to death (Trauth et al., 1989; Yefenof et al., 1993) or to their ability to inhibit cell traffic (Zahalka et al., 1993), cell-cell interactions (Zahalka et al., 1995; 25) or extravasation (Ruiz et al., 1993; Akiyama et al., 1995).
Recently it has been shown that MAbs can exert anti-tumor activity in other ways, e.g. by inhibiting metastases (Qi et al., 1995), tumor cell-substrata interactions (Guo et al., 1994), or tumor cell extravasation (Edward, 1995). In addition, it has been reported, that some MAbs can signal growth arrest and/or apoptosis of tumor cells, by acting as agonists (“negative signaling”) (Ghetie et al., 1992; Ghetie et al., 1994; Vitetta and Uhr, 1994; Trauth et al., 1989; Page and Defranco, 1988; Bridges et al., 1987; Funakoshi et al., 1994; Beckwith et al., 1991; Schreiber et al., 1992; Scott et al., 1985). “Negative signaling” is herein defined as the inhibition of cell growth by cell cycle arrest or the induction of apoptosis (programmed cell death). Indeed, in the case of B cell lymphoma, there is compelling evidence that both anti-idiotype (Levy and Miller, 1990; Hamblin et al., 1980) and anti-CD19 MAbs (Ghetie et al., 1992; Ghetie et al., 1994) exert their anti-tumor activities predominantly, if not exclusively, by signaling growth arrest and apoptosis. Other MAbs which also have signaling properties include anti-Fas (Trauth et al., 1989), anti-CD40 (Funakoshi et al., 1994), anti-Class II MHC (Bridges et al., 1987), anti-Her-2 (Scott et al., 1991), anti-Ley (Schreiber et al., 1992) and anti-IgM (Vitetta and Uhr, 1994; Page and Defranco, 1988; Beckwith et al., 1991; Scott et al., 1985). Furthermore, negative signaling can sometimes be optimized by hypercrosslinking with secondary antibodies or by using “cocktails” of primary antibodies (Marches et al., 1996).
In the case of anti-CD19, only a small percentage of MAbs can deliver growth inhibiting signals to neoplastic B cells and these require the addition of very large (i.e. hypersaturating) concentrations of antibody (Ghetie et al., 1994). Unfortunately, these large concentrations of antibody can activate unwanted immune responses which clear the therapeutic antibodies from the system and reduce the efficacy of treatment. Clearly, it is desirable to eliminate these unwanted immune responses in order to increase the efficacy of treatment, but to date, the means to eliminate these responses is not available.
The therapeutic or diagnostic usefulness of a monoclonal antibody (MAb) is dependent upon several factors. The MAb must possess sufficient binding affinity and a relatively high avidity for an antigen. The avidity of a MAb is based on the valency of the antibody (and the antigen) and the quaternary arrangement of the interacting components. The physical size of the molecule is also an important limiting factor. Thus while hundreds of MAbs recognize tumor antigens, few MAbs have proven useful for the diagnosis and treatment of tumors and neoplastic diseases, because they are not specific enough, they fail to have sufficient affinity or avidity or are too large to reach their antigens.
Part of the difficulty in designing antibody conjugates which possess sufficient affinity and avidity for tumor antigens and are of an appropriate isotype or subclass to efficiently initiate cell cycle arrest or apoptosis is that few MAbs possess sufficient affinity and avidity to be useful. For example, IgG molecules are monomers and have low valency; and thus, they have low avidity. IgM molecules have a higher valency but their size limits their ability to penetrate tissue and reach the desired antigenic target.
Homoconjugates of IgG molecules have been designed which possess increased valency and thus have higher avidity and are better able to promote effector function (PCT application WO92/04053; Wolff et al., 1992; 1993; Caron et al., 1992). But these homoconjugates possess two or more Fc regions; and thus, they can still elicit undesired immune responses which reduce successful treatment of a tumor or neoplastic disease. And although increasing their valency results in increased avidity, the physical sizes of the homoconjugates are also increased which may limit or even prevent them from being able to physically reach target epitopes.
There is a clear and present need for therapeutic MAbs with enhanced avidity which do not produce unwanted immune responses and are relatively small molecules which are capable of wide biodistribution. The present invention provides such compositions as conjugates of MAbs which do not possess an Fc region and yet are surprisingly active at signaling cell cycle growth arrest and/or apoptosis. The compositions of the present invention provide the further improvements of being relatively smaller than conventional conjugates of IgG MAbs, and are thus capable of wider biodistribution, and producing fewer unwanted immune responses because they do not signal effector functions.